Laminated substrate

Abstract

Provided is a laminated substrate having excellent fluorescence enhancement characteristics.　The laminated substrate comprises: a plate-shaped substrate having a pair of main surfaces that face each other; a plurality of metal-based particles disposed on one main surface of the substrate, separated from each other in a direction parallel to the one main surface; and a protective layer that covers the surfaces of the metal-based particles, wherein the plurality of metal-based particles satisfy at least one of (1) and (2), and the surface shape of the protective layer conforms to the surface shape of the metal-based particles. (1) The average particle diameter of the plurality of metal-based particles is 200-1600 nm, the average height of the plurality of metal-based particles is 55-500 nm, and the aspect ratio defined by the ratio of the average particle diameter to the average height is 1-8. (2) Each of the plurality of metal-based particles is disposed on one main surface of the substrate such that the average distance from the adjacent metal-based particles is 1-1,000 nm, and the standard deviation of the average distance is 30 nm or less.

Description

Multilayer substrate

[0001] The present invention relates to a laminated substrate.

[0002] A technique for enhancing fluorescence by utilizing localized plasmon resonance of metal nanoparticles is known (for example, Patent Document 1). Patent Document 1 describes the use of a laminate including an island layer (2) composed of a plurality of metal particles and a spacer layer (5) covering the island layer in a photochemical fluorescence sensor.

[0003] Japanese Patent Application Publication No. 08-271431

[0004] However, even in the laminate described above, there is still room for improvement in the degree of fluorescence enhancement.An object of the present invention is to provide a laminate substrate having excellent fluorescence enhancement properties.

[0005] [1] A laminated substrate comprising: a plate-like substrate having a pair of main surfaces opposing each other; a plurality of metal-based particles arranged on one main surface of the substrate at a distance from each other in a direction parallel to the one main surface; and a protective layer covering the surfaces of each of the metal-based particles, wherein the plurality of metal-based particles satisfy at least one of the following (1) and (2): (1) an average particle size of the plurality of metal-based particles is 200 to 1600 nm, an average height of the plurality of metal-based particles is 55 to 500 nm, and an aspect ratio defined as the ratio of the average particle size to the average height is 1 to 8. (2) The plurality of metal-based particles are arranged on the one main surface of the substrate such that the average distance between adjacent metal-based particles is 1 to 1000 nm, and the standard deviation of the average distance is 30 nm or less. [2] The metal-based particle assembly multilayer substrate according to [1], wherein the coefficient of variation (CV value) of the thickness of the protective layer is 30% or less. [3] The metal-based particle assembly multilayer substrate according to [1] or [2], wherein the average thickness of the protective layer is 300 nm or less. [4] The metal-based particle assembly multilayer substrate according to [3], wherein the average thickness of the protective layer is less than 50 nm. [5] The metal-based particle assembly multilayer substrate according to any one of [1] to [4], wherein the protective layer is amorphous.

[0006] A laminated substrate having excellent fluorescence enhancement properties is provided.

[0007] 1 is a cross-sectional view schematically showing an example of a laminated substrate according to the present invention.

[0008] <Laminated substrate> (1) Layer configuration The laminated substrate according to the present disclosure comprises: a plate-like substrate having a pair of opposing main surfaces, a plurality of metal-based particles arranged on one surface of the substrate at a distance from each other in a direction parallel to the one main surface, and a protective layer covering the surface of each of the metal-based particles. A laminated substrate according to one embodiment will be described with reference to Figure 1.

[0009] Fig. 1 is a cross-sectional view schematically showing an example of a laminated substrate. The laminated substrate 100 shown in Fig. 1 comprises a substrate 10 having a pair of opposing main surfaces 10a, 10b, a plurality of metal-based particles 20 arranged on main surface 10a of substrate 10 while being spaced apart from one another in a direction parallel to main surface 10a, and a protective layer 30 covering the surface of each metal-based particle 20.

[0010] (2) Substrate The substrate 10 is plate-shaped. Plate-shaped means that it has a pair of opposing main surfaces 10a, 10b and side surfaces connecting the main surfaces, and the thickness, which is the distance between the main surfaces, is 1 / 5 or less of the maximum diameter of the main surfaces (for example, the diagonal length in the case of a rectangle). The thickness may be 1 / 10 or less, 1 / 20 or less, or 1 / 30 or less of the maximum diameter of the main surfaces. The area of ​​the main surfaces is larger than the area of ​​the other surfaces that make up the side surfaces.

[0011] There is no particular lower limit to the area of ​​the main surface, and it is, for example, 0.25 μm ２ The laminated substrate may be a long roll, and there is no particular upper limit to the area of ​​the main surface. ２ The thickness of the substrate 10 is not particularly limited, and is, for example, 10 μm to 10 mm, preferably 20 μm to 5 mm, and more preferably 30 μm to 1 mm.

[0012] The substrate 10 is preferably non-conductive. If the substrate 10 is conductive, electrons can be exchanged between the metal-based particles formed thereon via the substrate, which tends to reduce the plasmon resonance effect. Examples of non-conductive materials that make up the substrate 10 include mica, SiO ２ , ZrO ２Examples of the insulating material include inorganic insulating materials such as glass, and thermoplastic resins.

[0013] The substrate 10 may be a light-transmitting or optically transparent substrate, or may be non-light-transmitting (light-absorbing).

[0014] The substrate 10 may have a single-layer structure or a multi-layer structure.

[0015] (2) Metal-Based Particles The plurality of metal-based particles 20 are arranged on main surface 10a of substrate 10 at intervals from one another in a direction parallel to main surface 10a. It is preferable that the plurality of metal-based particles 20 are not arranged at intervals from one another in a direction perpendicular to main surface 10a. In other words, it is preferable that metal-based particles 20 are arranged two-dimensionally along main surface 10a.

[0016] 1, metal-based particles 20 are preferably in contact with main surface 10a of substrate 10. Metal-based particles 20 are preferably not provided on the other main surface 10b of substrate 10.

[0017] A plurality of metal-based particles 20 constitute a metal-based particle assembly layer 21. The plurality of metal-based particles 20 can constitute a plasmonic structure. A "plasmonic structure" refers to a structure capable of exhibiting plasmon resonance. Plasmons are compressional waves of free electrons generated by the collective vibration of free electrons in a structure. When the plurality of metal-based particles 20 are a plasmonic structure, for example, when the laminated substrate is applied to a sensor element, the intensity of light emission (such as fluorescence) from a light-emitting body that labels a substance to be detected can be enhanced. Therefore, the laminated substrate can be suitably used as a light-emission enhancing element for various sensor elements. Applying the laminated substrate to a sensor element can improve the sensitivity, quantitative accuracy, and / or reproducibility (stability) of the sensor element.

[0018] In order to form metal-based particle assembly layer 21 into a plasmonic structure, metal-based particles 20 are preferably made of a material capable of plasmon resonance in the ultraviolet to visible light region. A material capable of plasmon resonance in the ultraviolet to visible light region means a material that, when made into nanoparticles or an assembly thereof, exhibits a plasmon peak that appears in the ultraviolet to visible light region in absorption spectrum measurement by absorptiometry.

[0019] Examples of metal-based materials capable of plasmon resonance in the ultraviolet to visible light region include precious metals such as gold, silver, copper, platinum, and palladium; metals other than precious metals such as aluminum and tantalum; alloys containing a metal selected from the precious metals and metals other than precious metals; and metal compounds (metal oxides, metal salts, etc.) containing a metal selected from the precious metals and metals other than precious metals. Among these, precious metals such as gold, silver, copper, platinum, and palladium are preferred as metal-based materials capable of plasmon resonance in the ultraviolet to visible light region, and silver is more preferred from the viewpoints of being inexpensive and having small absorption (the imaginary part of the dielectric function at visible light wavelengths is small).

[0020] The plurality of metal-based particles 20 satisfy at least one of the following (1) and (2), and preferably satisfy both of them: (1) The plurality of metal-based particles have an average particle size D of 200 to 1600 nm, an average height H of 55 to 500 nm, and an aspect ratio AR defined as the ratio of the average particle size to the average height of 1 to 8. (2) The plurality of metal-based particles are arranged on one main surface of the substrate such that an average distance L between adjacent metal-based particles is within a range of 1 to 1000 nm, and the standard deviation LCV of the average distance is 30 nm or less.

[0021] Average particle size D of the plurality of metal-based particles 20 that make up metal-based particle assembly layer 21 is preferably within a range of 200 to 1200 nm, more preferably 250 to 500 nm, and even more preferably 300 to 500 nm. The average particle size of metal-based particles 20 is preferably selected appropriately depending on the type of metal-based material that makes up metal-based particles 20.

[0022] The average particle size D of the plurality of metal-based particles 20 is the average particle size of the 10 selected metal-based particles when 10 metal-based particles are randomly selected in an SEM image observed from directly above metal-based particle assembly layer 21 composed of the plurality of metal-based particles 20, five tangent diameters are randomly drawn within each metal-based particle image (wherein all of the tangent diameter lines can only pass through the interior of the metal-based particle image, and one of these lines must be the longest line that can be drawn and passes only through the interior of the metal-based particle), and the average value (hereinafter, this average value will also be referred to as the "average tangent diameter") is taken as the particle size of each metal-based particle. The tangent diameter is defined as the perpendicular line connecting the distance between two parallel lines tangent to the outline (projected image) of a metal-based particle ("Particle Measurement Technology," Nikkan Kogyo Shimbun, 1994, p. 5).

[0023] To explain the method for measuring the average particle size in more detail, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ , x ２ , x ３ , x ４ , x ５ , x ６ , x ７ , x ８ , x ９ , x １０ ), 10 random numbers from 1 to 960 (y １ , y ２ , y ３ , y ４ , y ５ , y ６ , y ７ , y ８ , y ９ , y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x １ , y １ ), (x ２ , y ２ ), (x３ , y ３ ), (x ４ , y ４ ), (x ５ , y ５ ), (x ６ , y ６ ), (x ７ , y ７ ), (x ８ , y ８ ), (x ９ , y ９ ) and (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y １ ), (x ２ , y ２ ), (x ３ , y ３ ), (x ４ , y ４ ), (x ５ , y ５ ), (x ６ , y ６ ), (x ７ , y ７ ), (x ８ , y ８ ), (x ９ , y ９ ) and (x １０ , y １０ ) is obtained. The above-mentioned average tangent diameter is then obtained for each of a total of 10 metal-based particle images that include the coordinate point, and the average particle diameter is then obtained as the average of the 10 average tangent diameters. If at least one of the 10 coordinate points that make up the 10 random number combinations is not included in a metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0024] Average height H of the plurality of metal-based particles 20 is preferably within a range of 55 to 300 nm, and more preferably 70 to 150 nm. Average height H of metal-based particles 20 is the average value of 10 measurements obtained by randomly selecting 10 metal-based particles in an AFM observation image of metal-based particle assembly layer 21 and measuring the heights of these 10 metal-based particles.

[0025] The aspect ratio AR of the plurality of metal-based particles 20 is preferably within a range of 2 to 8, and more preferably 2.5 to 8. The aspect ratio AR of the metal-based particles 20 is defined as the ratio of the average particle diameter D to the average height H (average particle diameter D / average height H). The metal-based particles 20 may be spherical, but preferably have a flat shape with an aspect ratio AR of greater than 1.

[0026] In the metal-based particle assembly layer 21, the plurality of metal-based particles 20 are each arranged such that the average distance L between adjacent metal-based particles (hereinafter also referred to as the "average inter-particle distance") is 1 to 1000 nm, but is preferably within the range of 1 to 150 nm. In this specification, "adjacent metal-based particles" means that the metal-based particles are adjacent in a direction parallel to the main surface 10a. By arranging the plurality of metal-based particles 20 at such an average inter-particle distance, it becomes easier to obtain strong plasmon resonance and the effect of extending the range of action of plasmon resonance can be further enhanced. The average inter-particle distance is more preferably 1 to 100 nm, more preferably 1 to 50 nm, and even more preferably 1 to 20 nm. If the average inter-particle distance is less than 1 nm, electron transfer based on the Dexter mechanism occurs between the particles, which is disadvantageous in terms of deactivation of localized plasmons.

[0027] The average interparticle distance is the average value of the interparticle distances of 10 metal-based particles when 10 metal-based particles are randomly selected in an SEM image observed from directly above metal-based particle assembly layer 21 composed of a plurality of metal-based particles 20, and the interparticle distance between each of the selected metal-based particles and adjacent metal-based particles is determined. The interparticle distance between adjacent metal-based particles is a value obtained by measuring the distances between all adjacent metal-based particles (the smallest distance between the surfaces of adjacent metal-based particles) and averaging these values.

[0028] To explain the method for measuring the average interparticle distance in more detail, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ ~x １０ ), 10 random numbers from 1 to 960 (y １ ~y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x １ , y １ ) to (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y １ ) ~ (x １０ , y １０ ) is obtained. Then, for each of a total of 10 metal-based particle images that include the coordinate point, the inter-particle distance between the metal-based particle and the adjacent metal-based particle is obtained, and then the average inter-particle distance is obtained as the average value of the inter-particle distances between the 10 adjacent metal-based particles. If at least one of the 10 coordinate points that make up the 10 random number combinations is not included in the metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0029] The standard deviation of the average interparticle distance is preferably 0.1 nm or more, more preferably 0.2 nm or more, and even more preferably 0.3 nm or more.

[0030] The standard deviation of the average interparticle distance is defined as follows. In an SEM image observed from directly above metal-based particle assembly layer 21 composed of a plurality of metal-based particles 20, one metal-based particle is first selected at random, and the interparticle distance between that metal-based particle and adjacent metal-based particles is determined. The interparticle distance between adjacent metal-based particles is the average value obtained by measuring the distances between all adjacent metal-based particles (the smallest distances between surfaces). In the SEM image, nine metal-based particles different from the one selected at random are selected at random, and the interparticle distance between these nine metal-based particles is determined in the same manner as above. The standard deviation of the interparticle distances between adjacent metal-based particles for a total of 10 metal-based particles obtained in this manner is defined as the standard deviation of the average interparticle distance.

[0031] To explain more specifically the method for measuring the standard deviation of the average interparticle distance, first, an SEM observation image is measured using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. or an equivalent device. Next, the obtained observation image is read in 1280 horizontal pixels x 960 vertical pixels using free image processing software "ImageJ" manufactured by the National Institutes of Health. Next, 10 random numbers (x) are generated from 1 to 1280 using the random number generation function "RANDBETWEEN" in the spreadsheet software "Excel" manufactured by Microsoft Corporation. １ ~x １０ ), 10 random numbers from 1 to 960 (y １ ~y １０ ) are obtained. From each of the 10 random numbers obtained, 10 sets of random number combinations (x １ , y １ ) to (x １０ , y １０ The random numbers generated from 1 to 1280 are used as the x coordinates, and the random numbers generated from 1 to 960 are used as the y coordinates. １ , y １ ) ~ (x １０ , y １０) is obtained. Then, for each of a total of 10 metal-based particle images that include the coordinate point, the interparticle distance between the metal-based particle and the adjacent metal-based particle is obtained, and the standard deviation of the average interparticle distance is then obtained as the standard deviation of the interparticle distances between the 10 adjacent metal-based particles. If at least one of the 10 coordinate points that make up the 10 sets of random number combinations is not included in a metal-based particle image, or if two or more coordinate points are included in the same metal-based particle, the random number combination is discarded, and random number generation is repeated until all 10 coordinate points are included in different metal-based particle images.

[0032] From the viewpoint of exciting plasmons with high efficiency, it is preferable that the metal-based particles 20 have a smoothly curved surface, and it is more preferable that the surface has a flat shape with a smoothly curved surface, but the surface may include some minute irregularities (roughness), and in this sense the metal-based particles may be amorphous.

[0033] The number of metal-based particles 20 contained in metal-based particle assembly layer 21 is typically 10 or more, and preferably 30 or more. By forming a metal-based particle assembly layer containing 10 or more metal-based particles, strong plasmon resonance and an extension of the range of action of plasmon resonance are likely to occur due to interactions between localized plasmons of the metal-based particles. The number of metal-based particles 20 contained in metal-based particle assembly layer 21 may be, for example, 50 or more, or even 1,000 or more, or even 10,000 or more. The number density of metal-based particles 20 in metal-based particle assembly layer 21 is preferably 7 particles / μm ２ More preferably, 15 particles / μm ２ That's all.

[0034] It is preferable that metal-based particle assembly layer 21 does not exhibit electrical conductivity as a layer, and it is more preferable that metal-based particles 20 constituting metal-based particle assembly layer 21 are each non-conductive with adjacent metal-based particles. If there are locations in metal-based particle assembly layer 21 where electrons can be exchanged between metal-based particles 20, the plasmon resonance effect tends to be reduced. Therefore, it is preferable that metal-based particles 20 are reliably spaced apart and that no electrically conductive material is present between metal-based particles 20. Metal-based particles 20 themselves may be electrically conductive.

[0035] That metal-based particle assembly layer 21 does not exhibit conductivity as a layer can be confirmed, for example, by the fact that when a pair of tester probes of a multimeter [tester (Hewlett-Packard Company's "E2378A")] is brought into contact with metal-based particle assembly layer 21 at a distance of 10 mm to 15 mm, and the range is set to "30 MΩ," the resistance value under the measurement conditions is 30 MΩ or greater, resulting in a display of "overload."

[0036] (3) Protective Layer The protective layer 30 covers the surface of each metal-based particle 20. It is preferable that the protective layer 30 is in direct contact with the surface of the metal-based particle 20. The protective layer 30 may have a single-layer structure or a multi-layer structure. Typically, a portion of the surface of each metal-based particle 20 (the lower surface side in FIG. 1 ) is covered by the substrate 10, and therefore the protective layer 30 can cover at least a portion of the exposed portion of the surface of the metal-based particle 20 that is not covered by the substrate 10, and it is preferable that the protective layer 30 covers all of the exposed portion of the surface of the metal-based particle 20 that is not covered by the substrate 10. It is preferable that the metal-based particle 20 is not covered by either the substrate 10 or the protective layer 30 and does not have a surface that is exposed to the outside.

[0037] The surface shape of protective layer 30 follows the surface shape of metal-based particles 20. "The surface shape of protective layer 30 follows the surface shape of metal-based particles 20" means that the surface shape of protective layer 30 is approximately parallel to the surface shape of metal-based particles 20 in the cross-sectional view shown in Figure 1. Figure 1 is a schematic diagram of a cross section perpendicular to main surface 10a of substrate 10.

[0038] More specifically, in the surface shape of the protective layer 30, it is preferable that the thickness T of the protective layer 30 measured in a direction perpendicular to the main surface 10a, i.e., the distance perpendicular to the main surface 10a between the interface 20a between the protective layer 30 and the metal-based particle and the surface 30a of the protective layer 30 opposite the substrate 10, is substantially uniform. The phrase "the thickness T of the protective layer is substantially uniform" means that the coefficient of variation (CV value) of the thickness of the protective layer, i.e., (standard deviation of the thickness T of the protective layer / average value of the thickness T of the protective layer) is 30% or less.

[0039] The standard deviation and average value of the thickness T can be determined as follows. Specifically, a microscope image of a cross section perpendicular to the main surface 10a of the laminated substrate is prepared using a scanning microscope or the like. The magnification of the microscope image is set so that 10 to 250 metal-based particles are included. In the microscope image, the length of a straight line on the main surface 10a is divided into 51 equal parts, and coordinates 1, 2, ..., 50 are obtained from the left end of the cross-sectional image. Starting from these points, a group of straight lines perpendicular to the main surface 10a are drawn, and the intersections between the straight lines and the interfaces between the metal-based particles 20 and the protective layer 30 (intersections 1-1 to 50-1) are obtained, and the intersections between the straight lines and the surface 30a of the protective layer 30 opposite the substrate 10 (intersections 1-2 to 50-2) are obtained. The distance between intersections 1-1 and 1-2 is defined as the protective layer thickness T1, and the distance between intersections 2-1 and 2-2 is defined as the protective layer thickness T2, and protective layer thicknesses T1 to T50 are obtained. The average value of these 50 values ​​is defined as the average thickness of the protective layer. The standard deviation of these 50 values ​​is defined as the standard deviation of the protective layer thickness, and (standard deviation of the protective layer thickness / average protective layer thickness) is defined as the coefficient of variation (CV value) of the protective layer thickness. Note that, in the microscopic image, if a straight line does not intersect with the metal-based particles, the thickness of the straight line is not measured.

[0040] From the viewpoint of increasing the uniformity of the thickness of the protective layer and increasing the emission enhancement factor, the coefficient of variation (CV value) of the thickness of the protective layer may be 20% or less, preferably 18% or less, and more preferably 15% or less.

[0041] The average thickness of the protective layer is not particularly limited, and may be, for example, 3 nm or more, 10 nm or more, 15 nm or more, 20 nm or more, 30 nm or more, or 40 nm or more. The thickness of the protective layer may be, for example, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, 80 nm or less, or less than 50 nm.

[0042] The surface roughness of the protective layer is not particularly limited, but the arithmetic mean roughness Ra of the surface of the protective layer may be 10 nm or more, 15 nm or more, or 20 nm or more, and may be 500 nm or less, 300 nm or less, 200 nm or less, or 100 nm or less.

[0043] Protective layer 30 may have a portion that contacts and covers a portion of main surface 10a of substrate 10 that is not covered with metal-based particles 20, as shown in FIG.

[0044] The material of the protective layer is preferably a non-conductive material, i.e., an insulating material. Examples of insulating materials include SiO ２ , SiN, TiO ２ , Al ２ O ３ , Si ３ N ４ and organic insulating materials such as resin materials (e.g., polystyrene, acrylic resin, epoxy resin, etc.). The protective layer may be composed of two or more materials. The protective layer may have a single-layer structure or a multi-layer structure.

[0045] The protective layer is preferably amorphous. The amorphous nature of the protective layer 30 can be confirmed by HAADF-STEM and electron diffraction patterns. When an electron diffraction pattern is obtained for the portion of the protective layer in a cross-sectional STEM image, the absence of periodic contrast derived from crystals indicates that the protective layer 30 is an amorphous layer.

[0046] The water contact angle of the surface of the protective layer is not particularly limited, but may be 40° or less, preferably 30° or less, and more preferably 20° or less. By reducing the water contact angle of the surface of the protective layer, it becomes easier to bring an aqueous solution into contact with the protective layer. The aqueous solution may be, for example, a treatment liquid containing a capture substance or a precursor thereof used to immobilize a capture substance on the surface of the protective layer, or a sample that may contain a substance to be detected and that is supplied to the surface of the protective layer on which the capture substance is immobilized.

[0047] Forming a protective layer is advantageous in the following respects: [A] When metal-based particles are used as luminescence enhancing elements for enhancing the intensity of luminescence from a luminescent material that labels a substance to be detected, if the luminescent material is in direct contact with the metal-based particles, quenching due to electron tunneling from the luminescent material to the metal-based particles may occur, reducing the enhancement effect. By providing a protective layer on the metal-based particles, the luminescent material and the metal-based particles can be reliably separated, thereby suppressing quenching. [B] The stability (oxidation resistance, etc.) and environmental stability (for example, light resistance, humidity resistance, heat resistance, etc.) of the metal-based particles can be improved.

[0048] The laminate substrate may have further layers on top of the protective layer.

[0049] (Effects) The laminated substrate according to this embodiment can exhibit the following features [a] and [b]. These features are thought to be manifested by interactions between localized plasmons exhibited by a plurality of metal-based particles 20. [a] The range of action of plasmon resonance exhibited by metal-based particle assembly layer 21 is wide. This, for example, can widen the range over which the plasmon-induced light emission enhancement effect extends, making it possible to enhance the light emission of light-emitting bodies located within, for example, a range of several hundred nanometers (e.g., 200 nm) from the surface of metal-based particle assembly layer 21. [b] Metal-based particle assembly layer 21 exhibits strong plasmon resonance. This, for example, can obtain a strong light emission enhancement effect.

[0050] With regard to the above [a], the layered substrate according to the present invention can enhance the light emission of a light-emitting element that is arranged at a position, for example, 10 nm or more, further several tens of nm (for example, 20 nm, 30 nm, or 40 nm) or more, and further still 100 nm or more or 200 nm or more away from metal-based particle assembly layer 21.

[0051] Regarding the above [b], the strength of the plasmon resonance exhibited by the laminated substrate is not simply the sum of the localized plasmon resonances exhibited by the individual metal-based particles at a specific wavelength, but is even stronger. In the laminated substrate, the individual metal-based particles interact with each other to produce strong plasmon resonance. It is believed that such strong plasmon resonance is produced by the interaction between the localized plasmons of the metal-based particles.

[0052] Generally, when the absorption spectrum of a plasmon structure is measured by absorptiometry, a plasmon resonance peak (hereinafter also referred to as "plasmon peak") is observed as the peak located on the longest wavelength side in the ultraviolet to visible light region. The strength of the plasmon resonance of the plasmon structure can be evaluated from the magnitude of the absorbance at the maximum wavelength of the plasmon peak. The greater the absorbance value, the greater the strength of the plasmon resonance tends to be. When the absorption spectrum of metal-based particle assembly layer 21 having the above-described predetermined structure is measured by the following absorptiometry, the absorbance at the maximum wavelength of the plasmon peak located on the longest wavelength side in the ultraviolet to visible light region can be 1 or more, or even 1.5 or more, or even about 2.

[0053] The absorption spectrum of the plasmon structure can be measured by absorptiometry. Specifically, the absorption spectrum can be measured by irradiating the back side (the opposite side to the metal-based particle assembly layer) of a substrate on which a metal-based particle assembly layer has been laminated with incident light in the ultraviolet to visible light region from a direction perpendicular to the substrate surface, and measuring the intensity I of the transmitted light in all directions that has passed through the metal-based particle assembly layer side, and measuring the intensity I of the transmitted light in all directions that has passed through the opposite side to the incident surface, when a substrate having the same thickness and material as the substrate of the measurement sample is irradiated with the same incident light from a direction perpendicular to the surface of the substrate on which a metal-based particle assembly layer has not been laminated. ０and are obtained by measuring them using an integrating sphere spectrophotometer. In this case, the absorbance, which is the vertical axis of the absorption spectrum, is calculated using the following formula: Absorbance = -log １０ (I / I ０ The absorption spectrum can be measured using a general spectrophotometer.

[0054] Furthermore, when measuring the maximum wavelength of the plasmon peak on the longest wavelength side in the ultraviolet to visible light region and its absorbance, an objective lens and a spectrophotometer may be used to narrow the measurement field and perform absorption spectrum measurement.

[0055] The laminated substrate according to this embodiment can increase the light emission enhancement factor. While the reason for this is unclear, the following may be considered: A major factor in long-range plasmons is thought to be the resonance of whispering gallery modes propagating along the surface of individual metal-based particles with those of adjacent particles. The whispering gallery modes propagating along the surface of metal-based particles conform to the surface shape of the protective layer. In other words, it is speculated that a protective layer covering the metal-based particles with a uniform thickness results in less disturbance in particle surface propagation and inter-particle propagation than a protective layer with an uneven thickness, making it easier for resonance between adjacent particles, which is a cause of long-range plasmons, to occur.

[0056] (Method for Manufacturing Laminated Substrate) The laminated substrate can be fabricated, for example, by the following method. First, a plate-shaped substrate 10 is prepared. The surface of the substrate can be degreased and cleaned in advance. The degreasing and cleaning of the substrate can be performed by an appropriate method depending on the material of the substrate, and examples thereof include cleaning using a liquid selected from organic solvents and water. Cleaning methods include immersion in the liquid, ultrasonic cleaning while immersed in the liquid, and wiping with a cloth (woven fabric, nonwoven fabric, etc.) impregnated with the liquid. The degreasing and cleaning may be a combination of cleaning steps using two or more liquids. For example, when the substrate is a glass substrate, the degreasing and cleaning may include ultrasonic cleaning using alcohols (methyl alcohol, ethyl alcohol, isopropyl alcohol, etc.), ultrasonic cleaning using ketones (acetone, methyl ethyl ketone, methyl isobutyl ketone, etc.), and ultrasonic cleaning using ultrapure water, in this order.

[0057] Next, a metal-based fine particle layer is formed on the main surface of the substrate. Examples of this method are the following [A] to [C]. [A] A bottom-up method in which a plurality of metal-based particles 20 are grown from minute seeds on the substrate 10. [B] A method in which a plurality of metal-based particles 20 are coated with a protective film made of an amphiphilic material having a predetermined thickness, and then this is formed into a film on the substrate 10 by the LB (Langmuir-Blodgett) film method. [C] Other methods include post-treating a thin film prepared by vapor deposition or sputtering, resist processing, etching, and a casting method using a dispersion liquid in which metal-based particles are dispersed.

[0058] The above method [A] preferably includes a step of growing metal-based particles at an extremely slow rate on substrate 10 adjusted to a predetermined temperature (hereinafter also referred to as a "particle growth step"). According to a production method including such a particle growth step, metal-based particle assembly layer 21 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance can be obtained with good control.

[0059] In the particle growth step, the rate at which the metal-based particles are grown on substrate 10 is preferably less than 1 nm / min, and more preferably 0.5 nm / min or less, in terms of average height growth rate. The average height growth rate referred to here can also be referred to as the average deposition rate or the average thickness growth rate of the metal-based particles, and is defined by the following formula: average height of metal-based particles / metal-based particle growth time. The definition of "average height of metal-based particles" is as described above. The metal-based particle growth time refers to the time from the start to the end of growth of the metal-based particles, and specifically refers to the supply time of the metal-based material. When metal-based particle assembly layer 21 is considered as a film, the metal-based particle growth time can also be referred to as the film formation time. When the method for growing the metal-based particles is a sputtering method, the metal-based particle growth time is the sputtering time.

[0060] The temperature of the substrate 10 in the particle growth process is preferably 100°C or higher and 450°C or lower, more preferably 200°C or higher and 450°C or lower, even more preferably 250°C or higher and 350°C or lower, and even more preferably 300°C or thereabouts (approximately 300°C ± 10°C).

[0061] By adjusting the average height growth rate, substrate temperature and / or metal-based particle growth time, it is possible to control the average interparticle distance and its standard deviation, average particle size, average height and aspect ratio of the plurality of metal-based particles 20 grown on substrate 10.

[0062] The pressure (pressure in the chamber of the apparatus) when growing the metal-based particles is not particularly limited as long as it is a pressure that allows particle growth, but is usually less than atmospheric pressure. The lower limit of the pressure is not particularly limited, but is preferably 0.5 Pa or more, more preferably 6 Pa or more, and even more preferably 10 Pa or more, so that the average height growth rate can be easily adjusted within the above range.

[0063] The specific method for growing metal-based particles on substrate 10 is not particularly limited as long as it allows particles to grow at an average height growth rate of less than 1 nm / min, and examples of such a method include sputtering and vapor deposition methods such as vacuum deposition. Among sputtering methods, direct current (DC) sputtering is preferably used because it allows a metal-based particle assembly layer to grow relatively easily and makes it easy to maintain an average height growth rate of less than 1 nm / min.

[0064] The sputtering method is not particularly limited, and examples thereof include direct current argon ion sputtering, in which argon ions generated by an ion gun or plasma discharge are accelerated by an electric field and irradiated onto a target. Other conditions in the sputtering method, such as the current value, voltage value, and substrate-target distance, are appropriately adjusted so that particles grow at an average height growth rate of less than 1 nm / min.

[0065] In order to obtain metal-based particle assembly layer 21 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance with good control, it is preferable to set the average height growth rate to less than 1 nm / min in the particle growth step, as well as the average particle size growth rate to less than 5 nm. However, when the average height growth rate is less than 1 nm / min, the average particle size growth rate will usually be less than 5 nm. The average particle size growth rate is more preferably 1 nm / min or less. The average particle size growth rate is defined by the following formula: average particle size of metal-based particles / growth time of metal-based particles. The definitions of "average particle size of metal-based particles" and "growth time of metal-based particles" are as described above.

[0066] In order to obtain metal-based particle assembly layer 21 having the above-mentioned preferred average particle size, average height, aspect ratio, average interparticle distance, and standard deviation of the average interparticle distance, it is preferable to appropriately adjust the metal-based particle growth time in the particle growth step while taking into consideration the above-mentioned preferred manufacturing conditions.

[0067] (Formation of Protective Layer) Next, a protective layer is formed on the surface of the metal-based particle. The method for forming the protective layer is not particularly limited, but from the viewpoint of forming a surface shape that follows the surface shape of the metal-based particle, a dry film formation method such as a vapor deposition method, a sputtering method, an ion plating method, a CVD method, an ALD method, etc., or a wet film formation method such as a spray coating method is suitable.

[0068] Although the sputtering method is not particularly limited, it is preferable to use a radio frequency (RF) sputtering method. Argon gas or the like can be used as the sputtering gas. In order to reduce the contact angle of water on the surface of the protective layer, SiO is sputtered on at least the outermost surface of the protective layer by a sputtering method. ２ It is preferable to form a film.

[0069] From the viewpoint of forming a surface shape that follows the surface shape of the metal-based particles, it is preferable to increase the energy of the composition (sputtered particles) sputtered from the target by using high output in RF sputtering, for example, the discharge output (power) is 200 W or more, preferably 500 W or more, more preferably 1000 W or more. By increasing the energy of the sputtered particles, it is possible to densify the film while improving the conformability to the underlying structure, and also to form the film at a high speed. RF sputtering is preferably performed in an inert gas atmosphere such as argon gas, and it is preferable not to add oxygen to the inert gas atmosphere.

[0070] <Sensor Element> The laminated substrate can be used as a sensor element to be mounted in a sensor device for detecting a substance to be detected. For example, the laminated substrate can be cleaned with a piranha solution or the like, and then a capture substance that specifically binds to the substance to be detected can be introduced into a predetermined position on the surface of the protective layer 30 (the surface opposite the substrate 10) using a photolithography method or the like, thereby forming a sensor element.

[0071] The sensor element can detect a substance to be detected, for example, as follows. The detection may be qualitative or quantitative, and refers to, for example, the identification or quantification of the substance to be detected. When excitation light is irradiated onto a labeled substance to be detected that has specifically bound to a capture substance possessed by the sensor element, the label, which is a light-emitting body, is excited. Then, the metal-based particle assembly layer of the sensor element, which is preferably a plasmonic structure, resonates with the excited light-emitting body, resulting in plasmonic emission enhancement. The substance to be detected can be detected qualitatively or quantitatively by detecting the light emitted from the excited light-emitting body using a detector. For example, the amount of the substance to be detected can be measured qualitatively or quantitatively by measuring the light emission intensity.

[0072] According to a sensor element including a laminated substrate according to the present invention, the laminated substrate preferably includes a metal-based particle assembly layer that is a plasmonic structure, and this allows for enhanced plasmon emission to be exhibited, thereby improving detection sensitivity and detection accuracy.

[0073] The detection target substance is a substance to be detected qualitatively or quantitatively, and specifically binds to a capture target substance. The detection target substance is not particularly limited, and examples thereof include nucleosides, nucleotides, nucleic acids, proteins, sugars, glycoproteins, lectins, viruses, cells, antibodies, and exosomes. A sensor element in which the detection target substance is a biologically derived or biologically related substance is also called a biosensor element.

[0074] Nucleic acid refers to a polymer (nucleotide chain) of nucleoside phosphate ester in which a purine base or pyrimidine base and a sugar are glycosidicly bonded, and includes oligonucleotides including probe DNA, polynucleotides, DNA (full length or fragments thereof) in which purine nucleotides and pyrimidine nucleotides are polymerized, RNA, polyamide nucleotide derivatives (PNAs), etc. Furthermore, a nucleoside is a compound in which a base and a sugar are glycosidicly bonded, a nucleotide is a compound in which a phosphate is bonded to a nucleoside, and both nucleosides and nucleotides are compounds containing a base.

[0075] The term "specifically bind" broadly refers to chemical bonds between substances, including non-covalent bonds, covalent bonds, and hydrogen bonds, and examples include interactions between protein molecules and electrostatic interactions between molecules.

[0076] The captured analyte can be detected by labeling the analyte with a luminescent label in advance and detecting the luminescence from this label. The label may be a labeling substance that specifically binds to a complex obtained by the specific binding between the capture substance and the analyte. The luminescent substance is a substance that emits light upon injection of excitation energy from excitation light. The principle of luminescence in the luminescent substance is not limited, and examples include fluorescence, phosphorescence, chemiluminescence, etc. Conventionally known luminescent substances can be used.

[0077] The capture substance is a substance that specifically binds to the analyte substance and functions to capture it. The capture substance is immobilized, for example, on the surface of the protective layer 30. The capture substance is, for example, a substance having a binding active group that can specifically bind to the analyte substance. Examples of the binding active group include a carboxyl group and a hydroxyl group that can electrostatically interact with the analyte substance. The capture substance is not particularly limited, and examples include nucleosides, nucleotides, nucleic acids, proteins, sugars, glycoproteins, etc.

[0078] Examples of the sensor device include biosensor devices such as DNA sequencers, DNA microarrays, virus sensors, ion sensors, plate readers (protein chips, sugar chain chips, lectin chips, etc.), microspectrometers, and glucose sensors.

[0079] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0080] Example 1 (1) Degreasing and Cleaning of Glass Substrate A soda glass substrate having a square shape with sides of 50 mm and a thickness of 0.7 mm was prepared. This soda glass substrate was subjected to a degreasing and cleaning treatment using isopropyl alcohol, acetone, and then ultrapure water.

[0081] (2) Formation of a Metal-Based Particle Assembly Layer Using a DC magnetron sputtering device under the conditions below, silver particles were grown extremely slowly on a soda glass substrate that had been subjected to a degreasing and cleaning treatment, thereby forming a metal-based particle assembly layer over the entire surface of the substrate.

[0082] Gas used: Argon Pressure inside chamber (sputtering gas pressure): 10 Pa Distance between substrate and target: 100 mm Sputtering power: 4 W Average grain size growth rate (average grain size / metallic particle growth time): 0.9 nm / min Average height growth rate (= average deposition rate = average height / metallic particle growth time): 0.25 nm / min Substrate temperature: 300°C Metallic particle growth time: 360 min

[0083] From the SEM image, the silver particles constituting the metal-based particle assembly layer had an average particle size, based on the above definition, of 335 nm, an average interparticle distance of 16.7 nm, and a standard deviation of the average interparticle distance of 27.8 nm. １０ pieces (approximately 25 pieces / μm ２ ) silver particles. Based on the results of AFM imaging using a Keyence VN-8010, the average height of the silver particles was determined to be 96.2 nm. Therefore, the aspect ratio of the silver particles (average particle size / average height) was calculated to be 3.48.

[0084] When a tester [multimeter (E2378A manufactured by Hewlett-Packard Company)] was connected to the surface of the metal-based particle assembly layer formed on the substrate to check its conductivity, it was confirmed that it had no conductivity.

[0085] (3) Formation of a protective layer A SiO 2 film was formed on the surface of the substrate on which a plurality of metal-based particles had been formed under the conditions of an argon atmosphere without oxygen introduction, a discharge output (electric power) of 1000 W, and a protective layer growth rate of 1.8 nm / min. ２ SiO as a protective layer by RF sputtering using a target ２ A substrate that is considerably larger than the target is rotated above the target to deposit the protective layer over a wide area on the substrate, and the growth rate of the protective layer described above is the average rate over the entire area of ​​the deposited protective layer.

[0086] (4) Evaluation of the Protective Layer (4-1) Coverage State of the Metal-Based Particle Assembly Layer by the Protective Layer and Average Thickness of the Protective Layer The laminated substrate was cut along a plane parallel to the layer stacking direction, and a cross-sectional image of the cut portion was observed using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd., to confirm that the protective layer covered the entire surface of the metal-based particles opposite the substrate. Furthermore, when the surface of the laminated substrate opposite the glass substrate was measured by X-ray photoelectron spectroscopy using the device and measurement conditions described below, no silver atom signal was detected, confirming that the protective layer covered the entire surface of the metal-based particles opposite the glass substrate. Furthermore, it was confirmed that the protective layer was formed not only on the surface of the metal-based particles but also on the surface of the substrate exposed between the metal-based particles.

[0087] (4-2) Standard Deviation and Average Value of Thickness T of Protective Layer The standard deviation and average value of the thickness T of the protective layer based on the above measurement method using a scanning electron microscope "JSM-5500" manufactured by JEOL Ltd. were 4.0 nm and 28.0 nm, respectively. The coefficient of variation (CV value) of the thickness of the protective layer, i.e., (standard deviation of the thickness of the protective layer / average thickness of the protective layer), was 14.3%.

[0088] (4-3) Arithmetic mean roughness Ra of the protective layer surface The arithmetic mean roughness Ra of the protective layer surface of the laminate substrate was measured using an AFM. Specifically, an AFM (Keyence VN-8000) was used to obtain a 5 μm × 5 μm uneven profile in contact mode on the protective layer side surface of the laminate substrate, and then the entire effective area was designated, and the arithmetic mean roughness Ra of the protective layer surface was output from a "3D measurement" analysis. The arithmetic mean roughness Ra of the protective layer surface in Example 1 was 20.1 nm.

[0089] (4-4) Water Contact Angle on the Surface of the Protective Layer The contact angle of ultrapure water on the surface of the protective layer of the laminated substrate was measured using a fully automatic contact angle meter DM-701 (manufactured by Kyowa Interface Science Co., Ltd.) The water contact angle on the surface of the protective layer of Example 1 was 15.6°.

[0090] (4-5) Crystalline state of the protective layer The cross-section of the protective layer was confirmed by STEM electron diffraction pattern analysis under the following measurement conditions using the following device. The absence of diffraction contrast due to crystalline structure confirmed that the protective layer was amorphous. (Device and measurement conditions) Device: ARM200F manufactured by JEOL Ltd. Measurement method: Electron diffraction pattern observation Acceleration voltage: 200 kV

[0091] (4-6) Evaluation of Luminescence Enhancement Factor of Laminated Substrate Example A A metal-based particle assembly layer similar to that of Example 1 was formed on a 0.5 mm thick soda glass substrate by growing silver particles under the same conditions as in Example 1. This metal-based particle assembly layer had the same particle shape and average inter-particle distance as in Example 1.

[0092] Immediately thereafter, a SiO film having an average thickness of 28.0 nm was formed under the same conditions as in Example 1. ２ A protective layer was formed. A rhodamine B solution was spin-coated on the surface of the protective layer at a rotation speed of 2000 rpm, and then dried to fix the rhodamine B dye, which is an illuminant, to the surface of the protective layer, thereby obtaining a photoexcited light-emitting element A. The rhodamine B solution was prepared by dissolving rhodamine B dye (Rhodamine 110, Exciton) in ethanol to a concentration of 0.15 mM. The standard deviation and average thickness T of the protective layer were 4.0 nm and 28.0 nm, respectively. SiO ２ The surface shape of the protective layer conformed to the surface shape of the metal-based particles. The coefficient of variation (CV value) of the thickness of the protective layer, i.e., (standard deviation of the thickness of the protective layer / average thickness of the protective layer), was 14.3%.

[0093] Reference Example A' A reference-type photoexcited light-emitting element A' was obtained in the same manner as in Example A, except that no metal-based particle assembly layer was provided.

[0094] Comparative Example B A comparative photoexcited light-emitting element B in which a rhodamine dye was immobilized was produced in the same manner as in Example A, except that the protective layer was formed by spin-coating an SOG solution. Specifically, an SOG solution was spin-coated onto the metal-based particle assembly layer to form a SiO ２A protective layer was laminated. The SOG solution used was an organic SOG material "OCD T-7 5500T" manufactured by Tokyo Ohka Kogyo Co., Ltd., diluted with ethanol. SiO ２ The surface shape of the protective layer did not conform to the surface shape of the metal-based particles and was generally smooth. The standard deviation, average value, and coefficient of variation of the thickness T of the protective layer were 16.1 nm, 35.3 nm, and 45.7%, respectively. The arithmetic mean roughness Ra of the surface of the protective layer was 5.2 nm, and the water contact angle of the surface of the protective layer (before the rhodamine dye was fixed) was 64.1°.

[0095] Comparative Reference Example B' A reference-type comparative light-excited light-emitting element B' was obtained in the same manner as in Comparative Example B, except that the metal-based particle assembly layer was not provided.

[0096] Next, the optical sensing device shown in Fig. 1 was constructed. A fluorescence spectrophotometer (product name: PMA-12, manufactured by Hamamatsu Photonics KK) was used as the detector D. In the optical sensing device shown in Fig. 1, excitation light L1 having a wavelength of 532 nm was irradiated onto the laminated substrate 100 using a light source. Emission light L2 was measured by the detector D after passing through a wavelength cut filter F that cuts light of the wavelength of the excitation light L1.

[0097] For the emission spectrum obtained by detector D, the integral value of the emission spectrum over the wavelength range from 550 nm to 950 nm was calculated.

[0098] (Evaluation of Emission Enhancement Effect) The integral of the emission spectrum in Example A was calculated when the integral of the emission spectrum in Reference Example A' was set to 1, thereby obtaining the emission enhancement factor of Example A. Furthermore, the integral of the emission spectrum in Comparative Example B was calculated when the integral of the emission spectrum in Comparative Reference Example B' was set to 1, thereby obtaining the emission enhancement factor of Comparative Example B. The emission enhancement factors were 51.1 times and 17.1 times, respectively.

[0099] 10... substrate, 20... metal-based particle, 21... metal-based particle assembly layer, 30... protective layer, 100... laminated substrate.

Claims

1. A laminated substrate comprising: a plate-like substrate having a pair of opposing main surfaces; a plurality of metal-based particles arranged on one main surface of the substrate at a distance from each other in a direction parallel to the one main surface; and a protective layer covering the surfaces of the metal-based particles, the plurality of metal-based particles satisfying at least one of the following (1) and (2), and the surface shape of the protective layer conforms to the surface shape of the metal-based particles. (1) The plurality of metal-based particles have an average particle size of 200 to 1600 nm, an average height of the plurality of metal-based particles of 55 to 500 nm, and an aspect ratio defined as the ratio of the average particle size to the average height of 1 to 8. (2) The plurality of metal-based particles are arranged on the one main surface of the substrate such that the average distance between adjacent metal-based particles is 1 to 1000 nm, and the standard deviation of the average distance is 30 nm or less.

2. The laminated substrate according to claim 1, wherein the coefficient of variation (CV value) of the thickness of the protective layer is 30% or less.

3. The laminate substrate according to claim 1 or 2, wherein the protective layer has an average thickness of 300 nm or less.

4. The laminate substrate according to claim 3, wherein the protective layer has an average thickness of less than 50 nm.

5. The laminate substrate according to claim 1 or 2, wherein the protective layer is amorphous.

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